Cancer is a disease in which abnormal cells divide uncontrollably and possess the ability to invade other tissues. Normal cells grow and divide in a controlled way to produce more cells as they are needed to keep the body healthy. In order to ensure this process proceeds without mishap there are various protective mechanisms in place to ensure that damaged DNA is repaired and not passed on to subsequent generations of cells. However, due to a number of causes including, environmental stresses, genetic factors and age among other things, damaged or mutated DNA is not eliminated or repaired and can go on to affect normal cell growth and division. Consequently, uncontrolled division of abnormal cells leads to cancer, the second greatest cause of mortality in the United States. One of the key molecules involved in this process of eliminating damaged or changed DNA is the tumor suppressor protein p53. Functional p53 plays an important role in protecting human health and preventing disease. When p53 status is compromised, the cell is left unable to manage the replication of cells with damaged DNA. Unfortunately 50% of all cancers harbor mutations within p53, resulting in compromised activity (14); therefore understanding the exact mechanisms of how normal p53 prevents tumor progression is absolutely crucial to define targets for chemotherapeutic development that can effectively restore p53 function. Previous results in our lab suggest that p53 signaling, in response to DNA damage, may involve sphingolipids. Sphingolipids are a class of bioactive lipids that are important regulators of cell growth and death (1,2). While the bioactive lipids ceramide and sphingosine act as pro-death molecules, sphingosine-1- phosphate (S1P) has been shown to stimulate proliferation and angiogenesis. The primary enzyme regulating the balance between these pro-death and pro-growth bioactive molecules is sphingosine kinase 1 (SK1). This role highlights the importance of understanding how SK1 is regulated as its activity may play a role in controlling cell fate (3,4). Recently research from our lab established a profound and novel connection between p53 and SK1 (11-13). In these studies it was demonstrated that induction of p53 results in loss of SK1 through proteolysis and this p53-induced loss of SK1 is critical for allowing p53-mediated suppression of thymic lymphoma, osteosarcoma, and other cancers in vivo as evidenced in studies using the combined p53/SK1 knock out mice. Thus, regulation of bioactive sphingolipid levels may be a key component in the p53 DNA damage response and the interaction of these pathways warrants further investigation. The goal of this project is to define the protease(s) involved in p53-dependent SK1 degradation. Through various cell studies using MCF7 breast cancer cells as well as Caspase 2 knockout mouse embryonic fibroblasts we have generated strong preliminary data implicating Caspase 2 as the protease responsible for p53-dependent SK1 degradation. Caspase 2 is the most evolutionarily conserved member of the caspase family but despite this, the physiological function of Caspase 2 has remained enigmatic and controversial (42). Confounding this issue is the lack of knowledge of bona fide substrates of Caspase 2 and the fact that the few substrates identified thus far have not clearly revealed the enzyme's function. Interestingly, recent work has implicated a possible tumor suppressor role for Caspase 2 although the exact molecular mechanisms are still unknown (25,33,44). In light of these data this proposal aims to 1) Establish that Caspase 2 is required for p53-mediated proteolysis of SK1, 2) Define mechanistically the role of Caspase 2 in the regulation of the SK1/S1P pathway, 3) Demonstrate the biological significance of deregulation of the p53/Caspase2/SK1/S1P. The research outlined in this proposal will provide invaluable mechanistic insight into the exciting connection between p53 and SK1 while also demonstrating that SK1 is critical downstream target for the tumor suppressive action of p53. In addition this work also aims to uncover a novel role for Caspase 2 in the regulation of sphingolipids and identify a new Caspase 2 substrate, SK1 that could explain the characterized tumor suppressive roles of this enigmatic caspase.